In the field of therapeutics the use of proteins and antibodies and antibody-derived molecules in particular has been constantly gaining presence and importance, and, consequently, the need for controlled manufacturing processes has developed in parallel. The commercialization of therapeutic proteins, requires they are produced in large amounts. For this purpose the protein is frequently expressed in a host cell and subsequently be recovered and purified, prior to its preparation into an administrable form.
Depending on the protein to be expressed the choice of host cell may be a mammalian host cell, frequently a CHO (Chinese hamster ovary) cell, or a bacterial host cell. In the first case, the protein is typically secreted into the culture supernatant which is recovered, and the solution is then processed for protein purification.
When the host cell is a Gram negative prokaryotic cell, an often preferred expression system involves the newly synthesized protein accumulating within and being isolated from the periplasmic space. In this case, once the desired level of protein expression has been achieved, it is the cells that are harvested and processed. The protein is then recovered by means of subjecting the harvested cells to a protein extraction process which involves releasing the protein from the periplasm into solution and subsequent removal of cell debris and other impurities. These steps of cell harvest to protein release are typically included in what is termed primary recovery. The resulting protein-containing solution is then processed for protein purification. Preferred Gram negative prokaryotic cells used for periplasmic expression are generally Escherichia coli strains or Pseudomonas fluorescens cells.
Recovery of the heterologous protein expressed in the prokaryotic host cell during the primary extraction from the periplasmic space has often been a challenge. In this respect, different methods have been described in the prior art. For example, U.S. Pat. No. 8,969,036 describes the use of heat treatment to facilitate the subsequent isolation of functional Fab′ fragments of antibodies. WO 2006/054063 describes the inclusion of a non-lysing treatment in combination with heat treatment at the primary extraction stage. WO 2005/019466 describes the inclusion of an interruption step after fermentation but prior to extraction. US 2013/0060009 describes the pH adjustment of samples prior to undergoing heat treatment during the extraction step.
However, another significant challenge is the high level of impurities present after primary extraction which increases the burden on subsequent purification steps and the overall efficiency of the entire purification process. These impurities may be in the form of cell debris, host cell proteins (HCP), DNA, or product related impurities such as truncated forms of the expressed product, aggregates or other modified forms such as deamidated, isomerized, oxidized or other conjugated forms. Of these, recombinant protein degradation products, also termed product-related impurities, are often the most difficult to remove during the heat extraction process and primary recovery given that they have very similar physico-chemical properties, such as melting temperature (Tm), to the target protein.
Although the resulting protein solution obtained after extraction will then be processed for protein purification, the level of purity of this solution impacts overall efficiency and costs associated to the purification steps, and therefore that of the total therapeutic protein production. This effect becomes even more apparent when considering large-scale protein manufacturing. In particular product-related impurities have an impact on the final product's quality profile, the control of which is particularly relevant for consistency across different batches.
As such there remains a need in the art for methods that improve impurity removal during protein extraction from cell culture.